Explaining differences in sensitivity to killing by ionizing radiation between human lymphoid cell lines.

We surveyed five human hematopoietic cell lines (HSB-2, MOLT-4, Reh, CEM, and HL-60) to determine whether any simple correlates with sensitivity to killing by gamma-irradiation might be revealed. The clonogenic survival gamma-ray dose-response curves for these cell lines cover a wide range of sensitivities. Consistent with previous results for murine hematopoietic cell lines, there was a clear correlation between the rapidity with which irradiation induced apoptosis and clonogenic radiosensitivity of a cell line, although the relationship between timing of apoptosis and radiosensitivity differed between human and murine cell lines. Flow cytometric determination of cell cycle distribution after irradiation showed that differences between human hematopoietic cell lines, in the rate of induction of apoptosis, were generally related to the functioning of cell cycle checkpoints. Whereas the rapidly dying and radiosensitive HSB-2 cell line underwent apoptosis from different points in the cell cycle, the more slowly dying cell lines showed a variety of cell cycle arrest profiles and initiated apoptosis after accumulation of cells in the G2 phase. The lag-phase between arrest in G2 and induction of apoptosis was comparable for MOLT-4, Reh, and CEM; however, HL-60 cells showed a markedly longer G2 arrest that correlated with their greater radioresistance. The results suggest that the total length of time available for DNA damage repair (irrespective of whether this time accrues as blockage in G1, S, or G2), prior to potential activation of apoptosis, is a critical determinant of radiosensitivity in human hematopoietic cell lines. Comparison of the p53 status of these cell lines suggested that mutations in the TP53 gene are contributing to the delay of induction of apoptosis seen in the more radioresistant cell lines. The sensitivity of MOLT-4 and HL-60 cells to killing by DNA-associated 125I decays was determined and was found to correlate with the relative sensitivity of these lines to gamma-irradiation. The highly localized deposition of energy by 125I decays argues that DNA damage is a potent initiator of apoptosis in these cell lines. The results presented suggest that differences in the radiosensitivity of the cell lines examined reflect differences in the rapidity of induction of apoptosis and that radiation-induced cell death in hematopoietic cells can be explained as a response to DNA damage.

Importance of DNA damage in the induction of apoptosis by ionizing radiation: effect of the scid mutation and DNA ploidy on the radiosensitivity of murine lymphoid cell lines.

PURPOSE: To study the effects of the murine scid mutation and DNA ploidy on the susceptibility of lymphoid cell lines to induction of apoptosis by ionizing radiation and thereby to determine whether DNA lesions are critical initiators of apoptosis. MATERIALS AND METHODS: Sensitivity to killing and rapidity of induction of apoptosis following y-irradiation or DNA-associated 125I decays were compared in pre-B and pre-T cell lines derived from wild-type mice and from mice homozygous or heterozygous for the scid mutation. Effects of differences in DNA ploidy on the same endpoints were studied using pseudo-diploid and -tetraploid clones of a murine pre-T cell line. RESULTS: Pairs of pre-B- and pre-T cell-derived lines that expressed wild-type p53 and underwent rapid interphase apoptosis after irradiation were identified. In both cases, the scid homozygous cell lines were more sensitive to killing, suggesting that DNA repair capability influences susceptibility to induction of apoptosis. Increasing DNA ploidy in a cell line that undergoes rapid interphase apoptosis produced a corresponding increase in the number of DNA lesions required to produce a lethal event; again consistent with DNA being the target for radiation action. CONCLUSION: DNA damage is an important, if not the sole, initiator of external beam ionizing radiation-induced apoptosis.

Increasing the susceptibility of the rat 208F fibroblast cell line to radiation-induced apoptosis does not alter its clonogenic survival dose-response.

Recent studies have suggested a correlation between the rate and incidence of apoptosis and the radiation response of particular cell lines. However, we found that increasing the rate of induction of apoptosis in the fibroblast line 208F, by transfecting it with human c-myc, did not lead to a change in its clonogenic survival dose-response for either gamma-irradiation or 125I-induced DNA damage. It was also found that expression of mutant (T24) Ha-ras in the 208F line appeared to decrease the level of apoptosis per mitosis after irradiation and inhibited the formation of nucleosomal ladders, but did not affect either the onset of the morphological features of apoptosis or the clonogenic survival dose-response of the cells to either gamma-irradiation or 125I-induced DNA damage. Our findings suggest that it may be incorrect to make predictions about the radiosensitivity of cells based only on knowledge of their mode of death.